Overexpression of the oncogene MYC is characteristic of most high-risk diffuse large B-cell lymphomas, and is essential for the survival of lymphoma models. However, only half of DLBCLs assigned to high-risk subgroups of germinal center-like (GCB)-DLBCL by gene expression profiling bear activating genomic rearrangements of MYC, and MYC rearrangements are even less frequent in another common high-risk subtype, ABC-DLBCL. There is currently a poor understanding of the mechanisms that lead to MYC transcriptional dysregulation in DLBCL in the absence of an activating genomic rearrangement between MYC and an immunoglobulin gene locus (MYC-IG). The overall objective of this proposal is to define novel mechanisms of MYC regulation by distal enhancers. Our long-term goal is to use this understanding to develop improved therapeutic strategies and / or predictive biomarkers for DLBCL patients. Our working hypothesis is that transcriptional activation of MYC required to sustain DLBCL is dependent on the cis-regulatory activity of a small number of essential distal enhancer modules, located within much larger ?super-enhancer? regions. These modules are located either within the 3? region of the MYC locus on 8q24 (in DLBCL without MYC rearrangement), or in a genomic rearrangement partner locus such as 3q27, which is among the most common non-IG MYC rearrangement partners. We will utilize high-throughput CRISPR-interference profiling to perform robust functional interrogation of complex, multi-modular ?super-enhancers? present in 8q24 and 3q27 in DLBCL cell lines that lack MYC rearrangements (Aim 1), or bear t(3;8)(q27;q24) rearrangements (Aim 2) respectively. Having identified the discrete distal enhancer elements that are required for MYC activation, we will utilize a complementary set of experimental approaches, including genetic deletion or mutation of enhancer sequences, chromatin immunoprecipitation, reporter assays, in vitro DNA-protein binding assays, and perturbation of trans factors and upstream pathways to characterize the regulation of these elements. Our preliminary data suggests that distinct enhancer modules within the 3? MYC enhancer region are essential for DLBCLs with different aberrations affecting trans factors that selectively bind the essential enhancer modules. These aberrations include diverse signaling and genetic events that activate NF-kB factors, or alternately, hyperactivation of the coactivator OCA- B and a set of synergistically acting developmental transcription factors. We will evaluate the extent to which MYC activation in the context of a t(3;8) rearrangement is dependent on ?hijacking? of MEF2B-regulated enhancer modules that are typically responsible for activation of the oncogene BCL6. Upon successful completion of the proposed research, we expect to rigorously define multiple distinct mechanisms by which enhancers and associated trans-factors can drive MYC transcriptional activation in DLBCL. Our innovative approach for identifying the key mechanisms underlying oncogene activation by complex multi-modular distal enhancers may serve as a model for similar investigations in a wide variety of cancer types.